Acquisition of Freezing Tolerance of Resurrection Species from Gesneriaceae, a Comparative Study

Resurrection plants have the unique ability to restore normal physiological activity after desiccation to an air-dry state. In addition to their desiccation tolerance, some of them, such as Haberlea rhodopensis and Ramonda myconi, are also freezing-tolerant species, as they survive subzero temperatures during winter. Here, we compared the response of the photosynthetic apparatus of two other Gesneriaceae species, Ramonda serbica and Ramonda nathaliae, together with H. rhodopensis, to cold and freezing temperatures. The role of some protective proteins in freezing tolerance was also investigated. The water content of leaves was not affected during cold acclimation but exposure of plants to −10 °C induced dehydration of plants. Freezing stress strongly reduced the quantum yield of PSII photochemistry (Y(II)) and stomatal conductance (gs) on the abaxial leaf side. In addition, the decreased ratio of Fv/Fm suggested photoinhibition or sustained quenching. Freezing-induced desiccation resulted in the inhibition of PSII activity, which was accompanied by increased thermal energy dissipation. In addition, an increase of dehydrins and ELIPs was detected, but the protein pattern differed between species. During recovery, the protein abundance decreased and plants completely recovered their photosynthetic activity. Thus, our results showed that R. serbica, R. nathaliae, and H. rhodopensis survive freezing stress due to some resurrection-linked traits and confirmed their freezing tolerance.


Introduction
The rare phenomenon of desiccation tolerance in the vegetative tissues of vascular plants is possessed by so-called resurrection plants. They have the unique ability to restore normal physiological activity after desiccation to an air-dry state [1]. Drought stress inhibits photosynthesis mainly due to stomata closure, thus leading to mesophyll limitations [2] and disruption of the balance of energy capture and utilization via carbon metabolism [3]. As a result, reactive oxygen species (ROS) are generated and oxidative stress occurs in plant cells, leading to serious damage to DNA, proteins, and lipids [4]. Moreover, dehydration causes cell wall shrinkage and following plasma membrane rupture. Thus, resurrection plants have to cope with mechanical, structural, oxidative and metabolic stresses [5]. They evolve different strategies to preserve the integrity of membranes, macromolecules, and photosynthetic machinery by minimizing ROS production in the cells. Downregulation of photosynthesis, enhanced thermal energy dissipation, rearrangement of cell structure, upregulation of antioxidant defense system, synthesis of compatible solutes, and different protective proteins are part of those strategies of plants to cope with drought stress [4][5][6].
In addition to their desiccation tolerance, two of the European resurrection gesneriads, Haberlea rhodopensis and Ramonda myconi, are also freezing-tolerant species, as they survive in contrast to F v /F m , the actual efficiency of PSII, Y(II) significantly decreased during CA and it was reduced by 23%, 40%, and 30% in H. rhodopensis, R. serbica, and R. nathaliae, respectively on the 28 November ( Figure 3A).  Maximal quantum efficiency (F v /F m ; (A)), vitality index (R Fd ; (B)) and excitation pressure (1 − qP; (C)) of H. rhodopensis, R. serbica, and R. nathaliae leaves during cold acclimation , freezing stress (30 November), freezing-induced desiccation (3 December-7 February) as well after recovery of plants (11 June) in ex situ environmental conditions. Values are given as mean ± SE. Data represent the mean of n = 6. Changes between plants were statistically compared. The same letters within a graph indicate no significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA. Table 1. Relative water content (RWC) of the leaves of H. rhodopensis, R. serbica, and R. nathaliae during cold acclimation , freezing stress (30 November), freezing-induced desiccation (3 December-7 February) as well after recovery of plants (11 June)  The changes in the ratio of chlorophyll (Chl) fluorescence decrease (F m − F s ) to the steady-state Chl fluorescence (F s ), R Fd permits a fast evaluation of the photosynthetic activity and has been shown to correlate with the CO 2 assimilation rate of leaves [25]. R Fd values strongly declined during CA and they were reduced by about 60% in three Gesneriaceae species ( Figure 2B). The high correlation coefficient of Pearson (r > 0.970) was determined for the changes in R Fd and Y(II), but R Fd was the most sensitive parameter to low temperatures. It should be mentioned that the decrease in the photosynthetic activity during CA was not due to changes in Chl content ( Figure 4A). The Chl (a + b) content of both Ramonda species was not significantly influenced until 28 November and some enhancement in its content was measured in H. rhodopensis leaves. In addition, a good correlation was observed between the changes in R Fd and stomatal conductance (g s ), measured on the abaxial leaf side (r > 0.820). Similar to our previous results on H. rhodopensis, we registered much higher g s values on the abaxial side of R. serbica and R. nathaliae leaves compared to the adaxial side ( Figure 5). The reduction in the photochemical activity of PSII during CA was accompanied by an increased amount of closed PSII reaction centers, estimated by the changes in 1 − qP or so-called excitation pressure ( Figure 2C). High negative correlation coefficient of Pearson (r > −0.940) was determined for the changes in Y(II) and 1 − qP. Excitation pressure is suggested to be a major prerequisite for inducing efficient dissipation of excess excitation energy, thereby protecting the PSII reaction center from overexcitation. The results on the partitioning of absorbed light clearly showed that both the quantum yield of light-induced non-photochemical fluorescence quenching, Y(NPQ), and the quantum yield of non-regulated heat dissipation and fluorescence emission, Y(NO) increased during CA ( Figure 3).  , and R. nathaliae (C) during exposure to cold acclimation (7-28 November), freezing stress (30 November), freezing-induced desiccation (3 December-7 February) as well after recovery of plants (11 June) in ex situ environmental conditions. Values are given as mean ± SE. Data represent the mean of n = 6; Changes between plants were statistically compared. The same letters within a graph indicate no significant differences assessed by Fisher's LSD test (p ≤ 0.05) after performing ANOVA.

Effect of Freezing Stress on Photosynthetic Efficiency
Exposure of plants to −10 • C (30 November) decreased the leaf RWC, being the strongest in H. rhodopensis (by 32%) and weakest in R. nathaliae (by 16%). Freezing stress led to a strong reduction in photosynthetic efficiency. The ratio F v /F m decreased by 29%, 49%, and 34% in H. rhodopensis, R. serbica, and R. nathaliae (Figure 2A), respectively, whereas Y(II) was reduced by more than 70% ( Figure 3) compared with those measured on 7 November; g s additionally declined ( Figure 5) and R Fd was strongly inhibited ( Figure 2B). In addition, the values of 1− qP increased more than 6 times ( Figure 2C). Enhanced excitation pressure was accompanied by a double increase in Y(NO) (Figure 3) mainly due to a significant reduction in F m ( Figure S2). On the other hand, freezing stress caused a decrease in Y(NPQ) values, which was weaker in H. rhodopensis and stronger in R. serbica.
Fast desiccation of plants was induced when the temperature declined to −10 • C (30 November). Their RWC was reduced to 50% on 3 December and reached about 20% on 12 December (Table 1). During freezing-induced desiccation, the photosynthetic activity was strongly inhibited resulting in suppressed Y(NPQ), and the main part of absorbed light energy was emitted as heat. In fact, the proportion of Y(NO) was more than 90% in desiccated leaves (Figure 3), when the 1 − qP reached the highest values ( Figure 2C). In addition to increased harmless dissipation of excess excitation energy that cannot be used for photochemistry, the carotenoid content increased by 40% at the beginning of desiccation (date 3 December) and was closed to that measured on 7 November in desiccated plants (7 February).
As might be expected for resurrection plants, their photosynthetic activity was fully restored after rehydration.

Dehydrins
Dehydrin abundance in the leaves of H. rhodopensis, R. serbica, and R. nathaliae during CA and freezing-induced desiccation was monitored by Western blot using specific antibodies raised against the conserved K-segment of the proteins. Dehydrins were present in all investigated samples and at least 10 dehydrin bands with apparent molecular weight between 65 to 12 kDa were detected, but the protein pattern differed among the three species-H. rhodopensis and R. serbica profiles were more similar, while that of R. nathaliae was distinct ( Figure 6). Cold acclimation (28 November

Early Light-Induced Proteins (ELIPs)
As well as dehydrins, we monitored the accumulation of ELIPs in the leaves of the three resurrection species during cold acclimation and freezing-induced desiccation by Western blot. We detected two major and several minor bands in almost all samples with apparent molecular weight between 14-19 kDa, 13-19 kDa, and 14-17 kDa in H. rhodopensis, R. serbica, and R. nathaliae, respectively (Figure 7). The protein pattern was very similar in all three species. The two major bands are positioned close to 15 kDa and ELIPs were presented in well-hydrated plants with high physiological activity on 7 November. Cold acclimation (28 November) did not change significantly the ELIP abundance in H. rhodopensis and R. nathaliae, while in R. serbica its content surprisingly decreased. Temperatures drop to −10 • C (30 November) did not affect ELIP abundance in R. nathaliae, while in H. rhodopensis and R. serbica new bands could be distinguished. Freezing-induced desiccation (3 December-7 February) increased ELIP content in all resurrection species and in fully desiccated plants new ELIP bands appeared. In the leaves of R. serbica and R. nathaliae, two of the newly expressed bands, around 17 and 17-19 kDa respectively, were characterized by very high abundance. In H. rhodopensis those bands were very weak. During the recovery (date 11 June) of H. rhodopensis and R. serbica, ELIP signals almost disappeared, while in R. nathaliae leaves two bands could be clearly detected.

Physiological Changes in Response to Cold Acclimation and Freezing-Induced Desiccation
The resurrection plants growing in mountainous areas are exposed to and must overcome various stress factors. Many studies have been performed on the desiccation tolerance of H. rhodopensis, R. serbica, and R. nathaliae; however, the investigations on their freezing tolerance are restricted only to H. rhodopensis [8,10,14]. In the present study, we explored the response of these endemic and relict plants from the Balkan Peninsula to low positive and subzero temperatures. Exposure of plants to freezing stress induced dehydration of plants and they survived the harsh conditions due to some resurrection-linked traits. Being homoiochlorophyllous, they retain the chlorophyll content and photosynthetic apparatus during desiccation, which allow their rapid recovery upon rehydration. But on the other hand, the high amount of Chl molecules could be a source of singlet oxygen production and oxidative damage. We did not determine significant changes in Chl content in both Ramonda species during exposure to low temperatures and following desiccation of plants ( Figure 4A). It decreased by about 18% only after prolonged exposure of dry H. rhodopensis leaves to low temperatures, which is in agreement with the changes in Chl content induced by drought stress [18,26]. In addition, the reduction of total chlorophyll content in desiccated R. serbica and R. nathaliae leaves about 15-21% and 11-13%, respectively, was reported [23,27]. For example, changes in Chl content were determined upon drying R. serbica from negligible [24] to 40% [28], which is most probably due to differences in the experimental conditions (light, temperature, humidity). Moreover, decline of δ-aminolevulinic acid dehydratase activity, which is part of the biosynthetic pathway of Chl, was reported in both Ramonda species during dehydration [29].
In addition to the upregulation of the antioxidant system, resurrection plants avoid oxidative stress during dehydration by leaf folding so that the hairy abaxial leaf side becomes exposed to the light and there is a strong reduction in leaf area ( Figure S1) and the downregulation of photosynthesis (Figures 2 and 3). Reversible leaf curling was observed during CA when the overnight temperature decreased to −2 • C. Despite the unchanged RWC during CA, the efficiency of PSII electron transport gradually decreases ( Figure 3) and it was significantly reduced before exposure of plants to −10 • C. Taking into account the linear correlation between changes in R Fd and CO 2 assimilation previously observed [25], the higher sensitivity of R Fd than Y(II) suggested that CO 2 assimilation was more sensitive to low temperatures. Similarly, it has been shown that the reduction of CO 2 assimilation was affected before photochemical activity under moderate water stress conditions [18,28]. Formerly, we have shown that the amount of both large and small subunits of the Calvin cycle protein Rubisco, RbcL, and RbcS, decreased as a result of cold and freezing temperatures and freezing-induced desiccation [8]. Thus, the higher decrease in CO 2 assimilation than electron transport with low temperatures may also be due to the effect of temperature on the activity of enzymes involved in CO 2 fixation. A significant reduction of g s measured on the abaxial leaf side was observed during CA of three Gesneriaceae species ( Figure 5). Interestingly, the values of g s measured in R. nathaliae on 7 November were much higher compared with H. rhodopensis and R. serbica. It could be related to the higher frequency of stomata observed in R. nathaliae than in R. serbica and their distribution on both sides of the leaves [27]. Comparison of photosynthetic activity of three resurrection species in CA state showed that low positive temperatures decreased the quantum efficiency PSII electron transport, Y(II), and R Fd in R. serbica more compared with H. rhodopensis and R. nathaliae (23-28 November, Figures 2 and 3). A higher sensitivity of photosynthetic activity in R. serbica compared with R. nathaliae to high temperatures (up to 40 • C) and during water deficit has been reported [23,27] and this was related to drier and warmer habitats of R. nathaliae [22].
The response of three desiccation-tolerant species to freezing stress was similar. Exposure of plants to −10 • C decreased their RWC and the inhibition of photosynthetic activity was close to that of drought-induced desiccation. Freezing stress led to photoinhibition of PSII as assessed by the decrease in F v /F m . The reduced F v /F m ratio was due to a drop in F 0 values and a particularly strong decrease in F m ( Figure S2). The most significant decline in F v /F m was observed in R. serbica. The significant inhibition of F v /F m during freezing-induced desiccation was mostly attributable to the strong decrease in F m accompanied by a moderate decrease in F 0 . Indeed, a high Pearson correlation coefficient was determined for the changes in Fm and F v /F m (r > 0.96). The decrease in F 0 indicates the transformation of PSII centers to highly efficient quenchers, converting excitation energy to heat [30], while photoinhibition or maintaining a high NPQ in the dark, as observed in wintering evergreens [31] can lead to decreased F m values. Our results indicate that the reduced Y(II), when RWC drop to about 20 % (12 December), was mainly due to a decrease in the proportion of open PSII centers (qP) as well as a decrease in their excitation capture efficiency (F v /F m ; Figure S3). Downregulation of photosynthesis of overwintering plants was considered an adaptive photoprotective strategy that lowers photosynthetic efficiency to prevent damage [32,33].
All changes in photosynthetic activity induced by freezing stress were reparable due to the involvement of defense mechanisms, such as high amounts of carotenoids and increased dissipation of excess excitation energy that cannot be used in photosynthesis. The carotenoid content increased during CA, reaching a maximum after exposure of plants to −10 • C and, despite some decline, it remained high during freezing-induced desiccation (Figure 4). Similarly, it has been shown that carotenoid content was not significantly changed in desiccated leaves of R. serbica and R. nathaliae [27,28].
The downregulation of the photosynthetic electron transport rate was accompanied by a significant enhancement in excitation pressure ( Figure 2) and enhancement in thermal energy dissipation (Figure 3). Our results showed that non-photochemical fluorescence quenching has a major role in preventing photoinhibition during CA. Y(NPQ) gradually increased during exposure of plants to low temperatures and was maximal in the CA state (28 November). It remained high in H. rhodopensis and R. nathaliae when the temperature dropped to −10 • C. NPQ requires a trans-thylakoid pH, the presence of zeaxanthin and conformational changes in the light-harvesting antenna [24,34,35]. Fernández-Marín et al. [7] showed that freezing can induce zeaxanthin synthesis even in the absence of light and the potential activity of the enzyme violaxanthin de-epoxidase occurred at −7 • C. During freezing-induced desiccation, when photosynthesis was significantly inhibited, a low rate of electron transport induced a decreased ∆pH across the thylakoid membranes, and the main part of excess excitation energy was dissipated as heat. In severely desiccated plants Y(NO) became the main mechanism of energy dissipation providing effective protection against overexcitation.

The Role of Protective Proteins for Acquisition of Freezing Tolerance
Vitrification of the subcellular milieu during drought-or freezing-induced desiccation strongly impacts the biochemical processes in the cells. Enhanced cumulation of stressinduced proteins, such as dehydrins and ELIPs, is one of the main strategies used by resurrection plants to protect the macromolecules in the cells from the detrimental effect of desiccation [5]. Dehydrins belong to group 2 of LEA proteins and they are disordered hydrophilic proteins that are highly expressed under drought and cold stresses [36,37]. Our results revealed the different protein pattern accumulation of dehydrins in H. rhodopensis, R. serbica, and R. nathaliae during cold acclimation and freezing-induced desiccation as that of R. nathaliae differed from that of the other two plants. Immunoblot signals showed that dehydrin expression could be constitutive and/or induced in the three investigated species. Accumulation of dehydrins around 24-28 kDa was monitored in both Ramonda plants under freezing-induced desiccation, while in H. rhodopensis the proteins the molecular weight was around 20-22 kDa. Similar results regarding H. rhodopensis were obtained in our previous study [10]. In the protein pattern of R. nathaliae could be distinguished a unique major band around 38 kDa in all samples investigated was not present in R. serbica and H. rhodopensis leaves. Four upregulated dehydrin bands between 24-28 kDa were monitored in desiccated leaves of R. nathaliae [38]. The close molecular weight of detected proteins could be a result of phosphorylation as posttranslational modification [39] or alternative start and stop codons [40] thus generating different proteins from one gene. Low molecular weight dehydrins (20 and 12 kDa) accumulated during desiccation and after recovery in H. rhodopensis thylakoids [10,41]. Although the exact physiological functions of dehydrins have not yet been elucidated, it was suggested that they play a crucial role in the acquisition of desiccation tolerance stabilizing cell macromolecules, and acting as ROS scavengers [42]. Recently, LEA proteins in R. serbica leaves were characterized [43]. Dehydrins were divided into two groups, DEH1 and DEH2, and they are characterized by a very highly disordered structure with a typical random coil conformation. More members of DEH1 were upregulated during desiccation compared to members of DEH2. In addition, the structure of K-, S-, and Y-segments were elucidated. The authors suggested a nuclear localization for some of the dehydrins identified.
Van Buren et al. [44] proposed that duplication of ELIP genes in the genomes of resurrection plants is related to the evolution of desiccation tolerance and the protection of chloroplasts from photooxidative damage. ELIP transcripts and proteins are highly expressed in the leaves of H. rhodopensis in response to desiccation and during the early hours after rehydration [10,39,41,[45][46][47]. Recently, we found that rehydration after freezinginduced desiccation enhanced the ELIP transcript abundance more strongly in leaves compared with rehydration after drought [41]. Our results suggest that ELIPs play a role in the acquisition of freezing tolerance in R. serbica and R. nathaliae. To our knowledge, this is the first time when ELIP proteins are reported in R. serbica and R. nathaliae. The highest ELIP abundance matched the lowest levels of PSII efficiency. It was suggested that ELIPs stabilize photosynthetic complexes via chlorophyll binding [48]. 200 tandemly duplicated ELIP genes were determined in the genome of C. plantagineum [49]. All three investigated resurrection species R. serbica, R. nathaliae, and H. rhodopensis, showed different protein profiles of ELIPs accumulation during cold acclimation and freezing-induced desiccation. This could be related to the different environmental conditions in their natural habitats and/or gene duplications. The ELIP protein pattern of H. rhodopensis is very similar to previously obtained results [10]. Interestingly, H. rhodopensis plants rehydrated after drought-and freezing-induced desiccation also showed different protein patterns of ELIP accumulation [41]. Recovery decreased the ELIP content in R. serbica and H. rhodopensis, and to a lesser extent in R. nathaliae. Although the pattern of ELIP accumulation is similar in the three plants, it was definitely species-specific.

Plant Material and Experimental Design
Haberlea rhodopensis Frivaldszky tufts were initially collected from the Rhodope Mountains and reproduced vegetatively under ex situ condition as previously described, Ramonda serbica Pančić plants were collected from the Sharri Moutains, near the city of Prizren

Determination of RWC
The RWC of leaves was determined gravimetrically by weighing them before and after oven-drying at 80 • C to a constant mass and expressed as a percentage of water content in dehydrated tissue compared with water-saturated tissues using the following equation: RWC (%) = (FW − DW)/(TW − DW) × 100, where FW-fresh weight, DW-dry weight, and TW-turgid weight. TW was measured on leaves maintained for 12-16 h at 4 • C in the dark floating on water.

Pigment Content Determination
One-hundred mg of leaves was homogenized with ten mL of 80% acetone and the homogenate was centrifuged at 5000× g for 20 min at 4 • C. The Chl a, Chl b, and total carotenoid contents were determined spectrophotometrically by measuring the absorbance at 663, 645, and 460 nm using Multiskan Spectrum (Thermo Fisher Scientific, Waltham, MA, USA). The pigment content was calculated according to the equations of Lichtenthaler [50]. The data were presented on a dry weight basis.

Stomatal Conductance
Leaf porometer SC-1 (Decagon Devices, Inc., Pullman, WA, USA) equipped with a desiccant chamber was used to measure the stomatal conductance on the adaxial and abaxial side of the leaves. To ensure accurate conductance reading the sensor head was calibrated before measurements. Measurements were always performed between 11 a.m. and 1 p.m. Six leaves were marked and measured throughout the experiment.

Chlorophyll a Fluorescence Induction
Chl a fluorescence induction was measured with a portable fluorometer PAM-2500 (Heinz Walz GmbH, Effeltrich, Germany). The leaves were dark-adapted for 15 min and PAR of 90 µmol (photon) m −2 s −1 was used for the measurements. All used basic parameters were given by PamWin-3 software (Heinz Walz GmbH, Effeltrich, Germany). The maximum efficiency of PSII photochemistry was calculated as F v /F m immediately after the predarkening period. The actual efficiency of PSII electron transport during illumination was estimated at a steady state as Y(II) = (F m − F s )/F m [51]. The quantum yield of lightinduced non-photochemical fluorescence quenching was calculated as (Y)NPQ = (F m /F m ') − 1 and the quantum yield of non-regulated heat dissipation and fluorescence emission as Y(NO) = F s /F m [52]. All three yield parameters sum up to 1: Y(II) + Y(NPQ) + Y(NO) = 1. The ratio of the chlorophyll fluorescence decrease to steady-state fluorescence (used as a vitality index) was calculated as R Fd = F d /F s , where F d = F m − F s [25]. The excitation pressure of PSII, which gives an approximate measure of the reduction state of the first electron acceptor Q A of PSII, was calculated as 1 − qP, as qP is determined by the equation qP = (F m − Fs)/(F m − F 0 ) [53]. Six leaves from every plant species were marked and measured throughout the experiment.

Total Leaf Protein Extraction, SDS-PAGE, and Western Blot Analysis
Total leaf proteins were extracted in the sample buffer as described by Mihailova et al. [8]. The protein content was determined according to Bradford [54]. Isolated samples were separated on 12% (dehydrins) or 16% (ELIPs) SDS-PAGE (SE260 Mighty Small II, Hoefer, Holliston, MA, USA) according to Laemmli [55], modified by adding 8.0% glycerol to stacking and separating gels using a constant current of 20 mA per gel. Each lane contains 30 µg total leaf protein. Using semi-dry transfer (TE70X, Hoefer, Holliston, MA, USA) the proteins were blotted on nitrocellulose membrane for 90 min at a current of 1 mA cm −2 . Prestained protein standard (Precision Plus Protein™ Dual Color Standards, Bio-Rad, Hercules, CA, USA) was used for monitoring electrophoresis separation and transfer efficiency. Blots were probed with primary antibodies against ELIPs (AS06 147A, Agrisera, Vännäs, Sweden) and dehydrin K-segment (AS07 206A, Agrisera, Vännäs, Sweden). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was used (AS09 602, Agrisera, Vännäs, Sweden). The resulting bands were visualized by chemiluminescence, and signals were recorded on X-ray Blue films (Carestream Dental LLC, Atlanta, GA, USA). Films were scanned using an Epson Perfection V850 PRO scanner (Seiko Epson Corporation, Suwa, Japan).

Statistical Analysis
Leaves for biochemical analysis were sampled from five different tufts at each time point. Total leaf proteins were isolated two times and SDS-PAGE and immunoblot analysis were repeated twice. For Chl a fluorescence induction and stomatal conductance, the same six leaves were measured during the experiments. Comparison of means was made by the Fisher least significant difference (LSD) test at p ≤ 0.05 following ANOVA. A statistical software package (StatGraphics Plus, version 5.1 for Windows, The Plains, VA, USA) was used.
Pearson's correlation coefficient (r) was used to measure the strength of a linear association between two variables.

Conclusions
A comparison of the results obtained upon CA revealed the high resistance of investigated resurrection plants to low positive temperatures. They were able to maintain high photosynthetic activity and the main part of excitation energy was used for photochemistry. Plants keep their RWC during CA; thus, fresh leaves were exposed to subzero temperatures. Exposure of plants to −10 • C induced desiccation of plants and they survived the harsh conditions due to some resurrection-linked traits. Downregulation of photosynthesis, increased carotenoid content, and presumably enhanced dissipation of excess excitation energy protect plants against photooxidation. The protection of PSII during CA was mainly due to significant enhancement of non-photochemical quenching, whereas during freezing-induced desiccation the main part of absorbed light energy was emitted as thermal energy dissipation. Similar to H. rhodopensis, the accumulation of stress-induced proteins, such as dehydrins and ELIPs, suggests a role in the acquisition of freezing tolerance in R. serbica and R. nathaliae. Differences in the protein profile of ELIPs and dehydrins accumulation during cold acclimation and freezing-induced desiccation were observed between the three plant species. Regarding the photosynthetic activity, a stronger reduction in the quantum efficiency of PSII electron transport in R. serbica compared with R. nathaliae and H. rhodopensis was found only in the CA state, while the response of three desiccation-tolerant species to freezing stress was similar.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12091893/s1, Figure S1: Control, desiccated by freezing temperatures, recovered plants as well as a close-up view of H. rhodopensis, R. serbica, and R. nathaliae flowers. Figure S2: Minimum and maximum fluorescence levels of H. rhodopensis, R. serbica, and R. nathaliae leaves during cold acclimation, freezing stress, freezing-induced desiccation as well after recovery of plants in ex situ environmental conditions. Figure